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Avian infectious bronchitis virus IBV-K136, monoclonal antibody cell line 3D5 prepared by using avian infectious bronchitis virus IBV-K136, monoclonal antibodies, and applications of avian infectious bronchitis virus IBV-K136 and monoclonal antibodies

An IBV-K136, monoclonal antibody technology, applied in virus/phage, antiviral immunoglobulin, microorganism-based methods, etc., can solve the problem of few IBV antigen detection methods

Active Publication Date: 2015-08-19
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently established ELISA methods are mainly for IBV antibodies, and there are very few detection methods for IBV antigens. This technical bottleneck needs to be broken through

Method used

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  • Avian infectious bronchitis virus IBV-K136, monoclonal antibody cell line 3D5 prepared by using avian infectious bronchitis virus IBV-K136, monoclonal antibodies, and applications of avian infectious bronchitis virus IBV-K136 and monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 (screening of monoclonal antibody cell line 3D5 and preparation of monoclonal antibody)

[0023] 1.1 Antigen preparation

[0024] Chicken infectious bronchitis virus K136 strain was inoculated into 10-day-old SPF chicken embryos, and the allantoic fluid of chicken embryos was aseptically harvested 6 days later. The specific method of differential centrifugation of the allantoic fluid is as follows: After melting the virus liquid frozen at -20°C, centrifuge at 5000g at 4°C for 30min, discard the precipitate and take the supernatant, then centrifuge at 14000g at 4°C for 20min, discard the impurity protein, and take supernatant. Then perform ultracentrifugation: centrifuge the supernatant obtained in the previous step at 66100g (TYPE60Ti, BeckmanL8-M) at 4°C for 1.5h, discard the supernatant, and add 2mL TEN buffer solution (50mM Tris-HCL, 50mM NaCL, 5mM NaCL, 2 EDTA, PH7.4), and drop the precipitate under sterile conditions to make it disperse and dissolve. P...

Embodiment 2

[0037] Example 2 (identification of monoclonal antibody characteristics)

[0038] 2.1 Determination of monoclonal antibody ascites titer

[0039] Using the method of indirect ELISA, the prepared ascites was diluted 1:1000 first, and then diluted by 2 times, and then added to the enzyme-labeled wells coated with antigens, and the other steps were the same as before. Results The monoclonal antibody titer of this strain was 1:6.4*10 4 .

[0040] 2.2 Identification of monoclonal antibody subclasses

[0041] Follow the method introduced in the monoclonal antibody subclass kit instructions. The specific method is as follows: Add four kinds of monoclonal antibody ascitic fluid 1:5000 to dilute 100uL / well in the microtiter plate, incubate at 37°C for 1 hour, wash with PBST 3 times, each time for 5min; add working concentration of goat anti-mouse IgG respectively 1 , IgG 2a , IgG 2b , IgG 3 , IgM, IgA subclass serum 100 μL / well, add two wells of each subclass to each monoclonal ...

Embodiment 3

[0053] Embodiment 3 (preparation of chicken infectious bronchitis virus antigen diagnostic kit)

[0054] Using the monoclonal antibody prepared in Example 1 as a raw material, a chicken infectious bronchitis virus antigen diagnostic kit was prepared using the known technology in the field of antigen detection kit preparation technology. The diagnostic principle of the kit is the ELISA method.

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Abstract

The present invention provides avian infectious bronchitis virus IBV-K136, a monoclonal antibody cell line 3D5 prepared by using the avian infectious bronchitis virus IBV-K136, monoclonal antibodies, and applications of the avian infectious bronchitis virus IBV-K136 and the monoclonal antibodies. According to the present invention, the IBV-K136 strain is an endemic nephropathogenic infectious bronchitis virus strain and is obtained through screen, the IBV-K136 strain is adopted as antigen to amplify and then animal immunization is performed, the immune sera and myeloma cells are fused to screen hybridoma cells, the excellent performance avian infectious bronchitis virus monoclonal antibody cell line 3D5 is screened by using the method, and the cell line 3D5 is culture so as to express a lot of monoclonal antibodies; and the monoclonal antibodies prepared by using the process have characteristics of high monoclonal antibody titer, rapid antigen-antibody reaction, and strong specificity, and is especially suitable for further preparation of the avian infectious bronchitis virus antigen diagnosis kit, and the corresponding detection methods can be the double-antibody sandwich ELISA or colloidal gold method, and the like.

Description

technical field [0001] The invention relates to the technical field of antibody engineering, in particular to a strain of chicken infectious bronchitis virus IBV-K136, a monoclonal antibody cell line 3D5 prepared therefrom, a monoclonal antibody and applications thereof. Background technique [0002] Avian Infectious Bronchitis (IB) is an acute, highly contagious viral respiratory disease caused by Avian Infectious Bronchitis Virus. It is characterized by coughing, tracheal rales, and sneezing in sick chickens, and chicks are the most susceptible. Although attenuated infectious bronchitis vaccines and inactivated vaccines are widely used, due to the large number of serotypes of the disease, the weak cross-protection between types and the continuous emergence of new strains, it is difficult to prevent and treat it, which seriously threatens the The development of my country's chicken industry. [0003] Chicken infectious bronchitis virus (Infectious Bronchitis virus, IBV) be...

Claims

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Application Information

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IPC IPC(8): C12N7/00C07K16/10C12N5/20G01N33/577G01N33/569C12R1/93
Inventor 陈冰李亚杰杨保收郁宏伟梁武
Owner TIANJIN RINGPU BIO TECH
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